Cloning, Expression and Identification of Specific Genes related Osseointegration to Titanium.

本研究的目的是阐明钛诱导的细胞行为，并分离促进钛种植体-骨整合的特定基因。特别是，为了寻找与钛的骨整合相关的关键基因，将成骨细胞系MC3T3-E1细胞培养在钛镀膜玻璃上，并对其基因表达进行了分析。1)钛和铬镀膜玻璃在高真空条件下在抛光玻璃(直径33 mm×厚度1.5 mm)上镀膜钛和铬。将镀钛、镀铬的玻璃盘放置在6孔板中，用硅胶环固定。2)细胞贴壁、增殖和分化的检测：采用MTS法(美国Promega公司生产的CellTiter 96[0！R]水溶液)检测MC3T3-E1细胞的初始贴壁和增殖情况，用碱性磷酸酶活力法检测细胞分化程度。钛钴…表面贴壁细胞的平均相对数量种植后3h，TED玻璃数量显著高于未镀膜玻璃和镀铬玻璃。同样，镀钛玻璃在种植后第3天的平均细胞数显著高于其他条件。三种条件下种植的成骨细胞的平均碱性磷酸酶(ALP)活性均随时间的延长而升高，但在种植后14d镀钛玻璃上的ALP活性水平显著高于其他条件。结果表明，与未镀钛和镀铬玻璃板相比，镀钛玻璃板促进了MC3T3-E1细胞的贴壁、增殖和分化。3)成骨细胞基因表达的影响。然后用消减杂交法鉴定差异表达基因。经核苷酸序列测定，共获得7个克隆，分别为XAB-2、SOD-1、Galectin-1、肌动蛋白相关蛋白2/3mRNA、RIKEN cDNA2210013021基因、EST601086505F1和Est 01439。镀钛玻璃上Galectin-1、Xab-2、SOD-1基因表达均高于未镀钛玻璃，提示镀钛玻璃更有利于成骨细胞的贴壁、增殖和分化，且Galectin-1、Xab-2、SOD-1基因表达上调可能与钛-骨整合有关。较少

The aim of this study is to clarify the cell behavior induced by titanium and to isolate specific genes that promote the titanium implant-bone integration. Especially, to identify the master key genes related osseointegration to titanium, the osteoblastic cell line, MC3T3-E1 cells were cultured on titanium coated glasses and the gene expression were analyzed.1)Titanium and chrome coated glassesTi and Cr were coated to polished glass surfaces (33 mm in diameter by 1.5 mm in thickness) in a high vacuum condition. Ti and Cr coated glass disks were placed in a 6 well plate and fixed by silicone rings. Non-coated glass disks were served as controls.2)Cell attachment, proliferation and differentiation assessments.Initial cell attachment and proliferation of MC3T3-E1 cells were estimated by MTS-assay (CellTiter 96【○!R】 AQueous One Solution, Promega, USA), and cell differentiation was evaluated by alkaline phosphatase activity assay. The mean relative amount of the attached cells on the Ti-coa … More ted glass was significantly higher than those on non-coated and Cr-coated glass at 3 hours after seeding. On the same way the mean cell number on Ti-coated glass at 3 days after seeding was significantly higher than those of other conditions. The mean alkaline phosphatase(ALP) activity of the seeded osteoblasts also increased with time in these three conditions, while the ALP activity level on the Ti-coated glass at 14 days after seeding was significantly higher than those of other conditions. These results suggest that, Ti-coated glass plate accelerated the cell attachment, proliferation and differentiation of the MC3T3-E1 cells, compared to the non- and Cr-coated glass plates3)Effect of gene expression of osteoblast on titaniumOsteoblasts were cultured on Ti coated, Cr coated and non-coated glass plates. And then differentially expressed genes were identified by cDNA subtractive hybridization. Twenty independent clones were isolated and by nucleotide sequencing of these clones, seven clones were identified including EST genes ; xab-2,sod-1,galectin-1,actin related protein 2/3 mRNA, RIKEN cDNA 2210013021 gene, EST 601086505F1, and EST 01439. And galectin-1,xab-2,sod-1 gene expression on Ti-coated glass were higher than non-coated, Cr-coated glass.These results suggest that Ti-coating is more advantageous for osteoblastic cell attachment, proliferation and differentiation than Cr or non-coating and galectin-1,xab-2 and sod-1 up-regulation could be related to the Ti-bone integration. Less